Effects of Calcium Binding and the Hypertrophic Cardiomyopathy A8V Mutation on the Dynamic Equilibrium between Closed and Open Conformations of the Regulatory N‑Domain of Isolated Cardiac Troponin C

Troponin C (TnC) is the calcium-binding subunit of the troponin complex responsible for initiating striated muscle contraction in response to calcium influx. In the skeletal TnC isoform, calcium binding induces a structural change in the regulatory N-domain of TnC that involves a transition from a closed to open structural state and accompanying exposure of a large hydrophobic patch for troponin I (TnI) to subsequently bind. However, little is understood about how calcium primes the N-domain of the cardiac isoform (cTnC) for interaction with the TnI subunit as the open conformation of the regulatory domain of cTnC has been observed only in the presence of bound TnI. Here we use paramagnetic relaxation enhancement (PRE) to characterize the closed to open transition of isolated cTnC in solution, a process that cannot be observed by traditional nuclear magnetic resonance methods. Our PRE data from four spin-labeled monocysteine constructs of isolated cTnC reveal that calcium binding triggers movement of the N-domain helices toward an open state. Fitting of the PRE data to a closed to open transition model reveals the presence of a small population of cTnC molecules in the absence of calcium that possess an open conformation, the level of which increases substantially upon Ca²⁺ binding. These data support a model in which calcium binding creates a dynamic equilibrium between the closed and open structural states to prime cTnC for interaction with its target peptide. We also used PRE data to assess the structural effects of a familial hypertrophic cardiomyopathy point mutation located within the N-domain of cTnC (A8V). The PRE data show that the Ca²⁺ switch mechanism is perturbed by the A8V mutation, resulting in a more open N-domain conformation in both the apo and holo states

Expression of MT-I and MT-II mRNA in the liver of wild type and MT-I/II^−/− mice after brain injury was quantified by RT-PCR.

<p>(<b>A</b>) MT-I mRNA expression showed its greatest increase at 1 DPI and 3 DPI in wild type mice. (<b>B</b>) MT-II mRNA was increased at 1 DPI in wild type mice but was at peak levels at 3 DPI. MT-I/II^−/− mice were unable to increase MT-I and MT-II mRNA levels to the same extent as wild type mice. Groups that share lower case letters are not significantly different from each other (for both graphs; n = 6–7, error bars = SEM).</p

Displacement curves for MT-IIA in MT-I/II^−/− mouse brain homogenate (A) and MT-I/II^−/− mouse liver homogenate (B).

<p>Displacement curves constructed in solutions with protein content of 0.01 mg/ml and 0.1 mg/ml are parallel to the standard curve constructed in PBS. Therefore, no matrix effects were observed at these concentrations, in these tissues (n = 3, error bars = SEM).</p

Corticosterone concentrations in plasma after cryolesion injury to the brain were assayed by RIA in wild type and MT-I/II^−/− mice (A).

<p>No significant differences were found between the mouse strains. There was a significant increase in plasma corticosterone after cryolesion injury and sham surgery to a similar extent. Sham surgery does not induce a significant change in hepatic MT-I or MT-II mRNA expression (<b>B</b>). Time points that share letters are not significantly different (n = 5, error bars = SEM).</p

Liver MT-I/II protein levels after cryolesion injury to the brain were assayed by UC1MT ELISA in wild type mice.

<p>Hepatic MT-I/II protein levels were not increased until 3 DPI and showed a further increase at 7 DPI. Groups that share lower case letters are not significantly different from each other (n = 7, error bars = SEM). signalling mechanism is involved.</p

Standard curve generated by the UC1MT competitive ELISA with 4-parameter logistic curve fitted.

<p>Each point shown is the average of 4 quadruplicate standards.</p

Cross-reactivity of the UC1MT antibody for MT-III was tested by direct ELISA.

<p>Comparison of the standard curves for MT-IIA (blue lines) and MT-III (red lines) demonstrate UC1MT has very little if any cross-reactivity for MT-III. Data are expressed as the mean of triplicate measurements (error bars = SEM).</p

The NEAT Domain-Containing Proteins of <i>Clostridium perfringens</i> Bind Heme

<div><p>The ability of a pathogenic bacterium to scavenge iron from its host is important for its growth and survival during an infection. Our studies on <i>C</i>. <i>perfringens</i> gas gangrene strain JIR325, a derivative of strain 13, showed that it is capable of utilizing both human hemoglobin and ferric chloride, but not human holo-transferrin, as an iron source for <i>in vitro</i> growth. Analysis of the <i>C</i>. <i>perfringens</i> strain 13 genome sequence identified a putative heme acquisition system encoded by an iron-regulated surface gene region that we have named the Cht (<b><i>C</i></b><i>lostridium perfringens</i> <b>h</b>eme <b>t</b>ransport) locus. This locus comprises eight genes that are co-transcribed and includes genes that encode NEAT domain-containing proteins (ChtD and ChtE) and a putative sortase (Srt). The ChtD, ChtE and Srt proteins were shown to be expressed in JIR325 cells grown under iron-limited conditions and were localized to the cell envelope. Moreover, the NEAT proteins, ChtD and ChtE, were found to bind heme. Both <i>chtDE</i> and <i>srt</i> mutants were constructed, but these mutants were not defective in hemoglobin or ferric chloride utilization. They were, however, attenuated for virulence when tested in a mouse myonecrosis model, although the virulence phenotype could not be restored <i>via</i> complementation and, as is common with such systems, secondary mutations were identified in these strains. In summary, this study provides evidence for the functional redundancies that occur in the heme transport pathways of this life threatening pathogen.</p></div

ChtD and ChtE recombinant proteins bind heme.

<p>(A) Each purified recombinant protein and a hemin only sample was analyzed by absorbance spectroscopy between wavelengths of 250 to 650 nm. The presence of heme bound to the proteins was determined by a Soret absorbance at ~400 nm. (B) Detection of heme by LC-MS analysis of each purified recombinant protein, a hemin only sample and Tris buffer as a control. A single charged heme species, indicated by the arrow (→), is predicted to have a mass of 616 kDa.</p

Expression of ChtD, ChtE and Srt is upregulated under iron-limited conditions.

<p>(A) Western blot of whole cell lysates of JIR325 grown under normal conditions (TSB) or under iron-limited conditions (TSB containing 100 μM 2,2-dipyridyl, TSB + Dp). The cell lysates were probed with polyclonal antibodies to ChtD (anti-ChtD), ChtE (anti-ChtE) and Srt (anti-Srt). Bound antibodies were detected with HRP-conjugated goat anti-rabbit IgG (Millipore). The protein marker sizes (in kDa) are indicated on the left of the blot. (B) RT-PCR of <i>chtD</i>, <i>chtE</i> and <i>srt</i> on RNA isolated from JIR325 grown to mid-logarithmic phase under normal (TSB) and iron-depleted (TSB + 150 μM 2,2-dipyridyl, TSB + Dp) conditions. The <i>chtD</i> (576 bp), <i>chtE</i> (230 bp) and <i>srt</i> (276 bp) gene regions were PCR amplified using the appropriate primer pairs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162981#pone.0162981.s002" target="_blank">S1 Table</a>). DNA standard sizes (kb) are stated on the right of the gel. Lanes: RT- with reverse transcriptase (cDNA); NRT- no reverse transcriptase (RNA); NC- no template control (dH₂0); DNA- JIR325 genomic DNA; L-PCR markers (Promega).</p